Prepare in a sterile tube:
template RNA:
total RNA 0.1-5µg
or poly(A)+ mRNA 10ng-0.5µg,
or specific RNA 0.01pg-0.5µg
primer:
oligo(dT)18 0.5µg
or random hexamer 0.2µg,
or sequence-specific 15-20pmol,
deionized water (nuclease free) up to 11µl.
Incubate the mix at 70℃for 5 minutes and chill on ice.
Add the following in the order indicated:
5X reaction buffer 4µl,
10mM 4 dNTP mix 2µl (1.0mM - final concentration),
ribonuclease inhibitor 20u,
deionized water (nuclease free) to 19µl.
Incubate at 37℃for 5 minutes. If random primer is used, incubate at 25℃for 5 minutes.
Add 40 units of M-MuLV Reverse Transcriptase . Incubate the reaction mixture, containing oligo(dT)18 or sequence-specific primer at 37℃for 60 minutes. If using random hexamer primer, incubate at 25℃for 10 minutes and then at 37℃for 60 minutes.
Stop the reaction by heating at 70℃for 10 minutes. Chill on ice.
Note
The synthesized cDNA can be amplified by the PCR (see Protocols for PCR using Taq and Pfu DNA Polymerases) without intermediate phenol/chloroform extraction or ethanol precipitation.
Reference
Gerard, G.F. and D'Alessio, I.M., Methods in Molecular Biology, 16, Humana Press, Totowa, N.J., 73-93, 1993.
